Immunoassays use the antigen-antibody reaction for specific detection of small concentrations of an analyte in complex media such as blood, serum, urine or food. For decades, immunoassays have been indispensable tools in clinical diagnosis, environmental and food analysis for the detection of smallest concentrations of hormones, metabolites, proteins, biomarkers, toxins, pesticides, pathogenic bacteria and viruses. Many of the most important clinical-diagnostic analytes are only inexpensive and quickly measurable with immunoassays as there are no alternative analytical chemical methods offering an equivalent combination of high sensitivity (detection of nanomolar or lower concentrations) and high specificity (detection of the analyte in the presence of interfering substances with a chemically similar structure). Alternative methods such as chromatography (e.g., HPLC, gas chromatography), require an often multi-step sample preparation, e.g., by extraction and chemical derivatization.
Since the invention of the immunoassay 50 years ago by Rosalyn Yalow and Salomon Berson, a plurality of different embodiments have been developed which are designated as different immunoassay formats. The basic distinction between heterogeneous formats such as the ELISA (enzyme-linked immunosorbent assay), and homogeneous formats is made on the basis of the aggregate state of the antibody employed for detection: In the heterogeneous assays, the antibody (more rarely the antigen) is immobilized on a surface (reaction tube, microtiter plate, test strip). All the reaction steps such as washing and separation steps such as the detection likewise take place on the surface. In the homogeneous formats, none of the binding partners is immobilized, no washing and separation steps are necessary, and the detection likewise takes place in solution.
Another possibility to distinguish arises from the detection method used. As it is not possible to make the antibody-antigen binding reaction directly visible, one of the binding partners has to be chemically coupled with a molecular marker or “label” (exception: mass-sensitive biosensors based on surface plasmon resonance, grating couplers, quartz crystal microbalance, surface acoustic waves and similar techniques directly measure the molecular binding reaction in real time if one of the binding partners is immobilized on the sensitive surface. However, these methods are so far only employed in research applications, not in routine analysis). A suitable label has to have a high “specific activity,” i.e., it has to produce as many signaling events as possible per label molecule. The most frequently used labels include radioactive isotopes (radioimmunoassay, “RIA”), fluorescent dyes (fluorescein, rhodamines, etc.), fluorescent semiconductor nanoparticles (“quantum dots”), polymer nanoparticles (“latex beads,” agglomeration immunoassay), and enzymes, such as peroxidase, together with a colorimetric, fluorogenic or chemiluminescent enzyme substrate (ELISA). Radioactive isotopes possess the highest specific activity, even allowing for the detection of a single label molecule. Due to health hazards emanating from radioactivity, the associated high laboratory requirements (“isotope laboratories”) and high cost of the disposal of the residues, radioactivity immunoassays are increasingly replaced by alternative methods such as the ELISA.
Another distinction between different immunoassays results from the type of antibody used. Antibodies are proteins of a complex structure having constant regions which determine the structure and are similarly composed in all the antibody classes and highly variable regions which form the antigen binding sites. The originally used polyclonal sera containing a plurality of antibodies with a variable specificity and affinity are replaced in many applications by monoclonal antibodies which only contain exactly one antibody entity (“clone”) with a well-defined specificity and affinity. In theory, monoclonal antibodies may be produced in any amount with identical properties. Furthermore, so-called “antibody fragments” were also used for some analyses which can be produced by enzymatic digestion from whole antibodies. Single-chain antibodies are recombinant antibody fragments which can be produced by genetic engineering methods. In principle, all types of antibodies and molecular binders derived therefrom may be used in the known immunoassay formats.
The best known clinical analytes that can be detected with immunoassays include hCG (pregnancy test), thyroid hormones, such as TSH (thyroid disorders), steroid hormones (endocrinology, fertility) and PSA (biomarker for prostate carcinoma).
In summary, it can be said that immunoassays based on monoclonal antibodies or polyclonal antisera are among the most important analytical chemical methods of biotechnology, clinical diagnosis, environmental and food analysis and have a high commercial value.
A plurality of methods to perform an immunoassay is described in the prior art. Most heterogeneous immunoassays require several manual processes such as, e.g., pipetting steps, sample dilution and washing steps which have to follow an exact time protocol. Thus, trained personnel and a laboratory specifically equipped for these processes are typically required to correctly perform such assays. The required detection devices (“ELISA reader,” microtiter plate reader, immunoassay analyzer) are expensive and not portable. Protocols to perform an ELISA test are described in U.S. Pat. No. 4,016,043A and U.S. Pat. No. 3,839,153A, for example. Those skilled in the art easily recognize that such complex analytical processes can only be performed by trained personnel and only in a suitable laboratory environment. Automation of such processes is very laborious and requires highly complex automatons.
The heterogeneous lateral-flow immunoassay format (“test strip” immunoassay) has become accepted for on-site use. U.S. Pat. No. 6,156,271A describes a modern variant of this assay format. That test runs by itself after addition of the sample solution and detection is effected on a purely visual basis (reading of the presence of one or two colored bands on the test strip by the user). It is obvious that such a reading method cannot result in quantitative results (i.e., precise measurements of concentrations), but only a yes/no statement or a semiquantitative statement (concentration is below/above a certain threshold) is possible.
It is known that in contrast, homogeneous immunoassays are particularly suited to realize fast, quantitative and fully automated immunoassays. In homogeneous immunoassays, the signal generation is carried out simultaneously with the binding reaction. Unlike the above-described heterogeneous immunoassays, homogeneous immunoassays mostly consist of only one or a few dosing operations, an incubation time and a detection step. In the ideal case, only the mixing of the sample and a ready-made reagent mixture is required before a final value can be measured (so-called “mix and measure” test). In the case of a 1:1 mixture, i.e., identical volume fractions of sample and reagent mixture, dosing is possible with the simplest of means and with high precision.
It can be easily seen that a homogeneous immunoassay also allows for the shortest possible analysis duration as the measurement is possible immediately after achieving the binding equilibrium between antibody and antigen.
A disadvantage of homogeneous immunoassays is that higher chemical synthesis expenditure is required to couple the binding reaction with the signal generation.
In U.S. Pat. No. 4,960,693A, synthesis of an antibody-enzyme fragment 1 conjugate is described such that the functional enzyme (“holoenzyme”) is only formed after binding of the antigenenzyme fragment 2 conjugate. Chemical synthesis of such conjugates is sterically challenging and not equally well-suited for all types of analytes. Furthermore, additional reaction time is required due to the coupling with the enzyme reaction which is why that principle was not accepted.
A more universal and chemically easier approach for low molecular analytes is the fluorescence polarization immunoassay such as described in, e.g., EP0200960A. In that process, a conjugate of the analyte (estriol) and a fluorescent dye, in most cases fluorescein, was synthesized. The measured solution was illuminated with linearly polarized excitation light. The emitted fluorescent light was not polarized (depolarized) as long as the conjugate was freely in solution. Only after binding to the antibody, the rotation velocity of the conjugate was limited to such an extent that the emitted fluorescent light was also polarized. Through this, the binding equilibrium was measured in real time. The disadvantage of that method is the high expenditure in terms of equipment for detection as a polarized monochromatic light source and two fluorescence detectors equipped with polarizing filters are required for detection. Therefore, that principle was only accepted in special laboratory applications, but not in routine diagnosis and for on-site use. Additionally, it is only suited for low-molecular analytes. An additional method is required for protein analytes.
A technically easier detection of low-molecular analytes was realized in the method of Sellrie et al., using a europium cryptate immunoassay (abbr.: EuCr) as an example (US2008199972A). In that case, an EuCr-fluorescein conjugate is synthesized wherein a linker as short as possible consisting of one to no more than three methylene groups may be present between the EuCr and the fluorescein. Besides the anti-EuCr antibody, an anti-fluorescein antibody which, after binding to fluorescein, quenches the latter's fluorescence is additionally employed. The signal generation principle is based on the fact that for sterical reasons, only one of the two antibodies can bind the conjugate. The binding equilibrium and thus the fluorescence intensity depends on the concentration of free EuCr and can be measured directly in real time. An advantage of that system is that only one conventional fluorescence detector is required. The disadvantage is that it is likewise only suited for low-molecular analytes and the conjugate synthesis is chemically challenging.
An alternative homogeneous immunoassay method for low-molecular analytes likewise based on the principle of the binding-dependent fluorescence quenching was published by Coille et al. and in modified form by Tan et al. (I. Coille, S. Reder, S. Bucher and G. Gauglitz, Biomol. Eng 18 (2002), 273-280; Chongxiao Tan, Nenad Gajovic-Eichelmann, Walter F. M. Stocklein, Rainer Polzius, Frank F. Bier, Analytica Chimica Acta 658 (2010), 187-192). In that case, the analyte, tetrahydrocannabinol, is coupled to a protein, bovine serum albumin, which additionally carries several fluorescence quencher molecules on the surface. The anti-tetrahydro-cannabinol antibody is conjugated with a fluorescence molecule. The fluorescence is quenched when the antibody binds to the conjugate. If the analysis sample contains free tetrahydrocannabinol, the antibody binds the latter and fluoresces again. The advantage of that method is that, as with Sellrie et al., the simple measuring setup can be used for fluorescence measurement. Again, the chemically challenging synthesis of the analyte-quencher conjugate and the antibody-fluorophore conjugate is disadvantageous in that case.
Although the fluorescence measurement technique is very often employed in the biosciences, it is an elaborate and thus expensive measurement technique. Inexpensive and compact detectors, e.g., for on-site use, are rather realized with other detection methods. Electrochemical detection methods make the technically simplest and most strongly integrated measuring devices possible and have achieved acceptance, e.g., in the field of disposable biosensors for glucose over all the optical measuring methods. Therefore, efforts have been made for many years to realize homogeneous immunoassays based on a simple electrochemical detection principle.
A prerequisite for a homogeneous immunoassay with electrochemical detection is the availability of a sensitive electroanalytical method and a redox-active label/marker having high specific activity. Amperometric and voltammetric methods are sensitive and suitable methods. The specific activity of the redox label/marker mainly depends on the velocity constant of the heterogeneous electron transfer with the electrode and of the detection potential. Those skilled in the art have reached the consensus that a detection potential in the range of from −200 mV to +200 mV (against a silver/silver chloride reference electrode, in the following abbreviated with vs. Ag/AgCl) is ideal for measurements in biological solutions such as blood. The electron transfer constant is a function of the chemical structure of the redox label/marker as well as the diffusion coefficient and the electrode material used. A plurality of redox mediators are known which possess beneficial electrochemical properties and thus are in principle suitable labels/markers for an immunoassay. For use in biological media, the redox mediators must not react with sample constituents and have to be stable in an oxidized and reduced state and be sufficiently water-soluble. These include, e.g., the organic molecules such as hydroquinone/quinone, p-aminophenol, organic/inorganic sandwich molecules such as ferrocene, and inorganic complexes such as, e.g., hexacyanoferrate(II/III) or bis-bipyridine osmium. The water solubility of ferrocene and bis-bipyridine osmium is poor such that in most cases water-soluble derivatives are employed in this connection.
US2007/054317 describes water-soluble osmium-based redox molecules having the above-mentioned beneficial properties and different electrochemical immunoassay formats and electrode geometries, e.g., interdigital electrodes, by which the mentioned water-soluble redox molecules can be detected in a sensitive manner. Apart from the use of new redox mediators and of antibody and antigen conjugates based on the latter, the assay formats described in US2007/054317 do not surpass the prior art. In particular, no new, highly sensitive homogeneous immunoassay format is described.
EP1331482A1 describes an electrochemical immunoassay method wherein conjugates of ferrocene and other redox mediators with the analyte are employed. An enzyme biosensor, e.g., a glucose sensor, is used for detection, the release of the ferrocene-analyte conjugate leading to a modulation of the electrochemical signal of the glucose sensor. A disadvantage of the described coupling chemistry is that a protein such as human serum albumin, is used to produce a well water-soluble conjugate. It is known that it is not possible to synthesize protein-redox mediator conjugates with a precisely defined stoichiometric ratio. The characteristic of the test is thus always different from one batch to another. Those skilled in the art are also aware of the fact that such an indirect signaling method entails a danger that inhibitors from the enzyme reaction can modulate the enzyme reaction just like the redox mediator-analyte conjugate. A method in which the redox mediator conjugate directly modulates the electrochemical signal would be more robust.
The homogeneous electrochemical assay for hippuric acid in a microfluidic chip by Sung et al. is designed in accordance with such a principle (Sung Ju Yoo, Young Bong Choi, Jong Il Ju, Gun-Sik Tae, Hyug Han Kim and Sang-Hoon Lee. Analyst 134 (2009), 2462-2467). A ferrocene-hippuric acid conjugate is synthesized and an anti-hippuric acid antibody is used. The conjugate and the antibody bound to polymer particles are added to the sample in a precisely defined amount. If a sample contains no free hippuric acid, the conjugate binds to the antibody-loaded particles and is centrifuged with the latter. In a voltammetric experiment, a correspondingly small current will flow in the supernatant at the oxidation potential of the ferrocene. However, if the sample contains free hippuric acid, the particle-bound antibody preferably binds to the latter, the conjugate remains in solution during centrifugation and a correspondingly higher current will flow in the voltammetric experiment (at the oxidation potential of the ferrocene). That assay and all the assays following this principle have severe disadvantages which practically do not allow for a use in diagnostics.
First, the oxidation potential of ferrocene (+400 mV vs. Ag/AgCl) is too high for the selective detection in blood and blood serum. Second, a centrifugation step is not acceptable for most on-site applications. Third, despite the separation step (centrifugation), that method is very insensitive. For example, Sung et al. measured ca. 10 mg/mL of hippuric acid as the lowest concentration. That equals a concentration of 55 mM. However, typical immunoassay analytes have to be measured in the nanomolar concentration range (or lower), i.e., a million times more sensitive.
A homogeneous electrochemical assay which does without a separation step was presented by Di Gleria et al. (Katalin Di Gleria, H. Allen, O. Hill, Calum J. McNeil, Anal. Chem. 1988, 58, 1203-1205). A conjugate of ferrocene and the analyte, lidocain, as well as the anti-analyte antibody are also added to the sample in that case. As the detection principle, an enzyme reaction is used for which the ferrocenium-antigen conjugate is acting as a redox mediator. If the sample contains no free analyte, the conjugate binds to the antibody and the enzyme reaction, here glucose oxidase, is delayed and the amperometric current becomes smaller. A disadvantage of that format is that it is not sensitive enough for a typical immunoassay. For example, a lower detection limit of ca. 5 μM was achieved in serum, ca. 1000 times higher than in typical immunoassays.
A variant of that homogeneous redox immunoassay was described in which the redox mediator conjugate is directly electrochemically measured. In that case as well, only insufficient detection limits were achieved.
The assay and all the assays following a similar principle (i.e., the modulation of the diffusion constant through the antibody binding) have severe disadvantages which complicate use in diagnostics. The main disadvantage is low sensitivity as modulation of the diffusion constant only entails a slight signal modulation: even if the redox-active conjugate is completely bound by the antibody, an appreciable amperometric or voltammetric signal can still be measured. It would be desirable if the antibody-bound conjugate would produce no electrochemical signal at all.
The same applies to the principle of the modulation of an enzyme reaction through depletion of the redox mediator after binding to an antibody. That format could also only detect micromolar concentrations (or higher).
In summary, it can be said that homogeneous electrochemical immunoassays based on modulation of the diffusion constant of a low-molecular conjugate or modulation of an enzyme reaction through depletion of the redox mediator after binding to an antibody are not sufficiently sensitive to measure concentrations in the nanomolar range (or lower) as are typical for immunoassay analytes. Therefore, despite the inexpensive detection system, none of these assays can be used in a noteworthy commercial application.
Against this background, it could be helpful to provide a new improved method for performing an immunoassay in solution which no longer has the mentioned disadvantages and in particular offers a markedly improved sensitivity.